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Comprehensive Substrate Specificity Profiling of the Human Nek

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Comprehensive Substrate Specificity Profiling of the Human Nek RESEARCH ARTICLE Comprehensive substrate specificity profiling of the human Nek kinome reveals unexpected signaling outputs Bert van de Kooij1,2,3,4, Pau Creixell1,2,3,4, Anne van Vlimmeren1,2,3,4, Brian A Joughin1,2,3,4, Chad J Miller5†, Nasir Haider6, Craig D Simpson7, Rune Linding7, Vuk Stambolic6,9, Benjamin E Turk5, Michael B Yaffe1,2,3,4,8* 1Department of Biology, Massachusetts Institute of Technology, Cambridge, United States; 2Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, United States; 3Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, United States; 4MIT Center for Precision Cancer Medicine, Massachusetts Institute of Technology, Cambridge, United States; 5Department of Pharmacology, Yale School of Medicine, New Haven, United States; 6Department of Medical Biophysics, University of Toronto, Toronto, Canada; 7Biotech Research and Innovation Center, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark; 8Department of Surgery, Beth Israel Deaconess Medical Center, Divisions of Acute Care Surgery, Trauma, and Critical Care and Surgical Oncology, Harvard Medical School, Boston, United States; 9Princess Margaret Cancer Center, University Health Network, Toronto, Canada *For correspondence: [email protected] Abstract Human NimA-related kinases (Neks) have multiple mitotic and non-mitotic functions, but few substrates are known. We systematically determined the phosphorylation-site motifs for † Present address: Department the entire Nek kinase family, except for Nek11. While all Nek kinases strongly select for of Biochemistry, University of hydrophobic residues in the À3 position, the family separates into four distinct groups based on Washington, Seattle, United specificity for a serine versus threonine phospho-acceptor, and preference for basic or acidic States residues in other positions. Unlike Nek1-Nek9, Nek10 is a dual-specificity kinase that efficiently Competing interests: The phosphorylates itself and peptide substrates on serine and tyrosine, and its activity is enhanced by authors declare that no tyrosine auto-phosphorylation. Nek10 dual-specificity depends on residues in the HRD+2 and APE- competing interests exist. 4 positions that are uncommon in either serine/threonine or tyrosine kinases. Finally, we show that Funding: See page 26 the phosphorylation-site motifs for the mitotic kinases Nek6, Nek7 and Nek9 are essentially Received: 21 December 2018 identical to that of their upstream activator Plk1, suggesting that Nek6/7/9 function as phospho- Accepted: 13 May 2019 motif amplifiers of Plk1 signaling. Published: 24 May 2019 DOI: https://doi.org/10.7554/eLife.44635.001 Reviewing editor: Margaret C Frame, University of Edinburgh, United Kingdom Introduction Copyright van de Kooij et al. Protein phosphorylation by kinases plays an essential role in nearly all signaling events and physio- This article is distributed under logical processes within the cell. Protein serine/threonine kinases, together with ubiquitin ligases, the terms of the Creative play a particularly important role in mitosis, during which thousands of sites are phosphorylated Commons Attribution License, which permits unrestricted use (Dephoure et al., 2008). These mitotic phosphorylation events are predominantly mediated by the and redistribution provided that kinases Cdk1, Plk1, Aurora A and Aurora B, together with several members of the NimA-related the original author and source are kinase (Nek) family (Fry et al., 2017; Joukov and De Nicolo, 2018). For Cdk1, Plk1 and the Aurora credited. kinases, multiple substrates and detailed downstream signaling functions have been identified van de Kooij et al. eLife 2019;8:e44635. DOI: https://doi.org/10.7554/eLife.44635 1 of 30 Research article Biochemistry and Chemical Biology Cancer Biology (Joukov and De Nicolo, 2018), although much remains to be learned. In contrast, for the Nek family kinases only a few substrates have been identified, and many of their functions within the complex network of mitotic signaling are not well understood. The Nek kinase family consists of 11 serine/threonine kinases that together form an independent evolutionary branch of the human kinome (Figure 1A). The first human mitotic Nek kinase to be identified was Nek2, which is required for centrosomal disjunction following their duplication during S-phase, in order to assemble a bipolar mitotic spindle (Figure 1B; Fry et al., 2017) A similar func- tion was assigned to Nek5, which, in addition, also regulates centrosome composition in interphase (Prosser et al., 2015). Furthermore, Nek6, Nek7 and Nek9 have been shown to cooperate as a sig- naling module during various stages of mitosis (Belham et al., 2003). In this process, Nek9 is first phosphorylated in early mitosis by Cdk1, creating a binding site for the Plk1 polo-box domain. Plk1- mediated phosphorylation and activation of Nek9 then leads to Nek9-dependent phosphorylation and interaction-dependent activation of Nek6 and Nek7, allowing them to perform their down- stream functions (Roig et al., 2002; Belham et al., 2003; Yin et al., 2003; Richards et al., 2009; Bertran et al., 2011). As a signaling module, Nek6/7/9 have been reported to play a role in centro- some separation and maturation, nuclear envelope breakdown, metaphase and anaphase progres- sion, mitotic spindle formation and cytokinesis (Figure 1B; Roig et al., 2002; Yin et al., 2003; Yissachar et al., 2006; O’Regan and Fry, 2009; Salem et al., 2010; Bertran et al., 2011; Kim et al., 2011; Laurell et al., 2011; Sdelci et al., 2012; Cullati et al., 2017). Interestingly, although Nek6 and Nek7 share more than 80% sequence homology in their kinase domain, their mitotic functions are not completely redundant (Cullati et al., 2017). This is most likely due to their short but divergent N-terminal domains that drive differential interactions and might affect substrate selection (Vaz Meirelles et al., 2010; de Souza et al., 2014). Notably, the Nek kinase family is not exclusively involved in mitosis, but functionally diverse. Nek kinases have also been reported to play a role in meiosis, ciliary biology, and the response of cells to replication stress and DNA-damage (Figure 1B; Quarmby and Mahjoub, 2005; Melixetian et al., 2009; Moniz and Stambolic, 2011; Choi et al., 2013; Spies et al., 2016; Brien˜o-Enrı´quez et al., 2017). The functions of Nek3 and Nek4 are not clear but the current literature suggests that Nek3 may play a role in cell migration (Harrington and Clevenger, 2016), and Nek4 might be involved in regulating microtubule stability (Figure 1B; Doles and Hemann, 2010). We have previously determined the optimal substrate phosphorylation motifs, that is the sub- strate amino acid sequence preferentially phosphorylated by a given kinase, for the mitotic kinases Cdk1, Plk1, Aurora A, Aurora B and Nek2 (Alexander et al., 2011). This motif information strongly facilitates identification of kinase substrates and can help to understand the organization of complex phosphorylation networks like those in mitotic cells (Linding et al., 2007; Alexander et al., 2011; Kang et al., 2013). Therefore, to further catalog the optimal substrate motif-atlas for mitotic kinases and to characterize substrate-specificity of a complete and functionally diverse branch of the human kinome, we sought to determine the optimal phosphorylation-site motif for the Nek-kinases using Oriented Peptide Library Screening (OPLS) (Hutti et al., 2004), followed by direct experimental vali- dation together with molecular modeling. This revealed a surprisingly large amount of diversity in substrate-specificity among the Nek-kinases, and identified Nek10 as a dual-specificity kinase, that can phosphorylate substrates on tyrosine in addition to serine. Furthermore, these studies demon- strated that the mitotic kinases Nek6, Nek7 and Nek9, have a phosphorylation-site motif that is almost identical to the motif of Plk1, suggesting that the Nek6/7/9 module functions as a Plk1-motif amplifier. Results Determination of the phosphorylation-site motif for all members of the Nek kinase family To obtain the consensus phosphorylation-site motif for each individual Nek kinase, we performed Oriented Peptide Library Screening (OPLS) as described previously (Hutti et al., 2004; Alexander et al., 2011). In these experiments, each kinase is tested in 198 individual in vitro kinase reactions, each with a different peptide library as substrate (Figure 1—figure supplement 1A). Each peptide library is composed of a pool of 10-mer peptides containing an equimolar mix of serine and van de Kooij et al. eLife 2019;8:e44635. DOI: https://doi.org/10.7554/eLife.44635 2 of 30 Research article Biochemistry and Chemical Biology Cancer Biology A C Nek1 Nek3 Nek4 Nek5 -5-4-3-2-1 0 +1+2+3+4 -5-4-3-2-1 0 +1+2+3+4 -5-4-3-2-1 0 +1+2+3+4 -5-4-3-2-1 0 +1+2+3+4 P P P P TK G G G G A A A A TKL C C C C S S S S T T T T V V V V I I I I CMGC L L L L STE M M M M F F F F Y Y Y Y W W W W H H H H K K K K CK R R R R Q Q Q Q AGC N N N N D D D D E E E E pT pT pT pT CAMK pY pY pY pY R I R Y W M W F M Y L R R E MRI M M M Y F L I K FM Nek9 Nek6 L M W L W M QK Nek8 Nek7 MF RL F F F R F R pT KFK L MF KWF V V F Y I RI KR M HR F RK N F MK W Y F WW MD I I Y Nek2 norm. intensity) GL KVF WW YV MW L MRK L YpT L P Nek11 Nek10 2 LL WHpT C LM WH G FK W GK C LK QpT pT LpT pT Y FH NV YM I N L V L R LF R YY F LW C V Nek3 NY Q VR N Y V R RpT R E Y LV Nek4 H L Q Favored residues W RN pT W W pT
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